A bioterrorist arsenal is likely to include a broad spectrum of bacterial species including the Category A pathogens Yersinia pestis (plague), a Gram-negative species, and Bacillus anthracis (anthrax), a Gram-positive species. Broad-spectrum antibiotics are potentially the most valuable for biodefense because these agents can be used quickly even in the absence of confirmatory diagnostic identification of species and can be stockpiled more efficiently and at lower cost. The overall goal of this proposal is to identify new antibacterial agents useful for biodefense against a broad spectrum of species and a wide range of drug resistance mechanisms. We will focus on a novel chemical series that potently inhibits UDP-GlcNAc enolpyruvyl transferase (EPT), an essential, broadly conserved, bacterial-specific enzyme catalyzing the first committed step in peptidoglycan synthesis. We will optimize this inhibitor series through a focused application of rational and structure-based drug design to generate lead compounds that are effective in an in vivo infection model. The specific objectives are to (1) Lead validation: Optimize the potency of quinoxaline inhibitors on Y. pestis and B. anthracis EPT.; (2) In vitro/antimicrobial lead optimization: Demonstrate target specificity and inhibition of growth or viability of natural and engineered surrogates of Y. pestis and B. anthracis', and (3) Objective 3. In vivo lead optimization: Demonstrate in vivo efficacy of EPT inhibitors against surrogate priority pathogens in animal models of infection and optimize activity for preclinical candidate.